Cyclophosphamide monohydrate and lactose

ABSTRACT

A parmaceutical preparation consisting essentially of a lyophilized cake of cyclophosphamide monohydrate and lactose prepared by the process of freezing cyclophosphamide, lactose and water, lyophilizing part of the water, obtaining and melting a supersaturated cyclophosphamide-lactose solution, precipitating cyclophosphamide as a hydrated polymorph, refreezing the solution and lyophilizing the water not bound to cyclophosphamide or lactose.

This is a continuation of co-pending application Ser. No. 07/805,181,filed Dec. 10, 1991, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to the lyophilization of cyclophosphamide.

U.S. Pat. No. 4,537,883 to Alexander et al. (Mead Johnson & Co.)discloses various lyophilizates of cyclophosphamide. These lyophilizatesare prepared by lyophilizing a solution of cyclophosphamide and one ormore excipients and re-hydrating the product such that it contains about4% moisture. The patent is based upon a comparative study oflyophilizate cakes and the dissolution time for lyophilizates ofcyclophosphamide prepared using a number of excipients. The studyconcludes that the lyophilizate prepared with mannitol gives a bettercake and faster dissolution time than the lyophilizates prepared withother excipients. The patent also teaches that the lyophilizedcyclophosphamide-mannitol composition exhibits better thermal stabilityif it contains an equimolar amount of water based on cyclophosphamide.The preferred lyophilizate contains 20 parts cyclophosphamide, 1.25 to 2parts water and 10 to 85 parts mannitol. Among the excipients evaluatedin the patent are mannitol, sodium bicarbonate, lactose, polyvinylpyrrolidone (PVP), arginine, and tartaric acid.

In lyophilizing cyclophosphamide, it is known that cyclophosphamidemonohydrate is more stable than anhydrous cyclophosphamide. As a result,a practice has developed wherein in preparing lyophilized pharmaceuticalpreparations containing cyclophosphamide, the bound water or water ofcrystallization is removed from the cyclophosphamide in the lyophilizerand a small amount of water is back added to the composition to convertthe less stable anhydrous cyclophosphamide product to the more stablemonohydrate. This practice is described in U.S. Pat. No. 4,537,883 toAlexander and in T.R. Kovalcik and J.K. Guillory "The Stability ofCyclophosphamide in Lyophilized Cakes," J. Paren. Sci. & Tech., 42, No.1, p. 29-37 (1988). It is also described in commonly assigned U.S.applications 07/597,965 and 07/583,896.

The process of back adding water is not satisfactory when using certainbulking agents, such as lactose, which take up the water which is backadded to the composition. Such bulking agents compete with thecyclophosphamide for the water. Because these bulking agents have ahigher affinity for water than cyclophosphamide, the cyclophosphamidemay never be converted to the monohydrate as desired.

Published European Application 0 401 894 to Pharmachemie discloses alyophilization process for preparing freeze-dried cyclophosphamidemonohydrate without any bulking agent. Cyclophosphamide monohydrate iscommercially available neat, i.e., without any bulking agent. Thiscompound might be administered to patients directly but it is difficultto dissolve and hence difficult to reconstitute. Accordingly, it wouldbe desirable to have a more readily dissolvable form of cyclophosphamidemonohydrate. While not desiring to be bound, it is believed thatlyophilizates in accordance with the invention described below may havea unique crystalline structure which renders them more easy to dissolveand reconstitute.

SUMMARY OF THE INVENTION

The present invention relates to a method for lyophilizingcyclophosphamide which overcomes the need to add water back to thelyophilizate to stabilize it and enables one to prepare lyophilizates ofcyclophosphamide monohydrate using bulking agents such as lactose whichhave a strong affinity for water.

The method of the present invention comprises the sequential steps of:

freezing a bulk solution containing cyclophosphamide, a bulking agent,and water;

removing a portion of the water from said frozen solution bylyophilization, said water being removed in an amount such that uponmelting said solution, a solution supersaturated with saidcyclophosphamide frozen solution is formed;

melting said solution to produce a solution supersaturated with saidcyclophosphamide and said bulking agent;

precipitating said cyclophosphamide as a hydrated polymorph;

refreezing said solution containing said precipitated cyclophosphamide;and

removing by lyophilization the free water in said refrozen solutionwithout removing water bound to said cyclophosphamide or said bulkingagent to yield a lyophilizate of cyclophosphamide monohydrate.

The present invention further provides a pharmaceutical preparationcomprising a lyophilized cake containing cyclophosphamide monohydrateand lactose. Previously, it was not believed to be possible to preparelyophilizates of cyclophosphamide monohydrate and lactose because it wasbelieved that water should be added back to the composition to form themonohydrate. Using this process, the lactose preferentially binds to thewater added back to the composition and a lyophilizate of the lessstable anhydrous cyclophosphamide is obtained.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 is a pressure and shelf temperature diagram for thelyophilization cycle used in the Example.

FIG. 2 is a pressure and product temperature diagram for thelyophilization cycle used in the Example.

DEFINITIONS

The term "nominal volume" or "nominal vial volume" as used herein meansthe vial volume as stated by the manufacturer exclusive of headspace,i.e., not the total actual volume of the vial.

DETAILED DESCRIPTION

In accordance with the invention, a cyclophosphamide bulk solution islyophilized in two stages to provide efficiently a storage stablecomposition without requiring further hydration to yield a stablelyophilized composition. In performing the lyophilization, thelyophilization conditions are controlled so that the cyclophosphamide isprecipitated as a hydrated polymorph and then the process is completedunder conditions which are carefully selected so that water bound to thecyclophosphamide as the monohydrate is not removed during the remainderof the lyophilization process. As initially precipitated,cyclophosphamide may have one or more molecules of water bound to it.For this reason, it is referred to as a "hydrated polymorph." The firstmolecule of water bound to the cyclophosphamide is believed to be morestrongly bound to the compound and more energy is required to removethis molecule of water than is required to remove other water moleculessubsequently bound to the compound.

The lyophilization process can be considered as occurring in two stages.In the first stage, a supersaturated solution of cyclophosphamide isproduced from which hydrated cyclophosphamide polymorph is precipitated.In the second stage, any remaining unbound or free water any water notbound to the cyclophosphamide as the monohydrate is removed. The secondstage procedure is more delicate than the first because water must beremoved without removing the water bound to the cyclophosphamide as themonohydrate.

The first stage lyophilization may be carried out under harsherconditions than the second stage, e.g., at higher temperatures and/orlower pressures to sublimate a major portion of water from the frozensolution to produce a supersaturated solution of cyclophosphamide uponmelting. In the second stage, care is taken not to generate theanhydrous cyclophosphamide by removing the water bound to thecyclophosphamide as the monohydrate. For this reason, the second stagemay require milder conditions.

In the first stage lyophilization the vials are cooled to an initialtemperature of -15° C. or less and more preferably -30° C. or less. Thevials are held at the temperature about 4 to 5 hours. When the solutionis completely frozen, the pressure is reduced and the condensor isactivated. Pressures less than 8000 micron, preferably less than 750microns and most preferably about 500 microns are used. The vials areheld under this condition about one hour. The shelf temperature is thenraised to about 0° C. and the vials gradually warm. As the vials warm,about 60 to 70% of the water is removed by the lyophilizer. This usuallyrequires about 10 hours. Throughout this disclosure, times for variousstages of lyophilization are sated. These times will vary depending upona number of factors including the capacity and operation of thelyophilizer, the number of vials, the dosage, fill level, the nature ofthe bulking agent, etc.

The preferred bulking agent is lactose, but mannitol, lower molecularweight amino acids such as glycine, valine, etc. and other bulkingagents described in U.S. Pat. No. 4,537,883 are also believed to beuseful. Amorphous bulking agents such as bicarbonate and sorbitol arenot believed to be useful in the process.

The bulk solution may contain about 2.5 to about 3.5% and preferablyabout 3.0 to 3.3% cyclophosphamide, and about 1 to 10% bulking agent.Amounts will vary with the selection of the bulking agent. For lactose,the bulk solution preferably contains about 3.0 to 3.3% (30-33 mg/ml)and most preferably 3% cyclophosphamide and 2 to 7% lactose (20-70mg/ml). Cyclophosphamide-lactose cakes produced in accordance with thepresent invention most preferably contain about equal amounts ofcyclophosphamide and lactose. While the amounts of lactose andcyclophosphamide disclosed above may be used, the ratio of lactose tocyclophosphamide generally ranges from about 0.3 to 1.7 parts lactoseper 1 part cyclophosphamide.

The bulk solution is easily prepared by dissolving the bulking agent andcyclophosphamide in distilled or deionized water at room temperature.The cyclophosphamide can be obtained from Farmos Company as thecrystalline monohydrate. The amorphous anhydrous cyclophosphamide couldalso be used to prepare the bulk solution.

The method of the present invention is generally practiced using 10 to100ml vials (nominal volume). The bulk solution is added to provide 100to 2000mg cyclophosphamide dosages as shown on the next table:

    ______________________________________                                                         Nominal                                                      Dosage           Vial Volume                                                                              Bulk Solution*                                    ______________________________________                                        100     mg       10 cc      3.3 ml                                            200     mg       20 cc      6.6 ml                                            500     mg       30 cc      17 ml                                             1       g        100 cc     34 ml                                             2       g        100 cc     68 ml                                             ______________________________________                                         *30 mg/ml cyclophosphamide                                               

The first stage lyophilization is stopped when a majority and preferablyabout 60% to 70% of the water is removed. Enough water must remain inthe vial after the first stage lyophilization to supply the water boundto the cyclophosphamide and the bulking agent and to dissolve thecyclophosphamide. In general, the amount of water in the vial should notbe reduced to less than about 30% of the total contents of the vial. Ifmore water than this is removed, incomplete conversion of thecyclophosphamide to the stable monohydrate may result.

The objective of the first stage lyophilization is to produce asupersaturated solution from which the cyclophosphamide precipitates asa hydrated polymorph on warming. When the desired amount of water hasbeen removed, the vacuum and condensor are deactivated and thelyophilization chamber is allowed to warm to a temperature at which thesolution melts and the cyclophosphamide precipitates from solution as ahydrated polymorph. This temperature is typically about 25° C. Anytemperature which melts the cake can be used. The term "precipitate" isused herein because it is not entirely clear whether the hydratedpolymorph is crystalline or not and hence it is not clear whether theterm "crystallize" is accurate although it is believed that the hydratedpolymorph is probably crystalline.

The second stage lyophilization can be carried out under the same ormilder conditions than the first stage. The second stage conditions areselected such that water bound to the cyclophosphamide monohydrate andthe bulking agent (some bulking agents bind water and other bulkingagents do not) is not removed in the second stage. Typical second stageconditions begin with shelf temperatures of about 0° C. to -30° C. Afterthe solution is completely frozen, the pressure is reduced to about 500to 8000 microns and the condensor is activated. The shelf temperature isthen raised to 0° C. and the cake is allowed to warm gradually to thattemperature. As explained earlier, these conditions will also varysomewhat with the nature of the bulking agent as well as the lyophilizercapacity, the vial size, the number of vials, etc. Once the cake reachesthe shelf temperature (about 2 hours), the process is usually complete.

The lyophilizate of the present invention will contain the bound water,the cyclophosphamide and the bulking agent. The amount of water in thelyophilizate will vary with the nature of the excipient since someexcipients will bind water and others will not. In the case of acyclophosphamide-lactose product containing equal amounts of lactose andcyclophosphamide, the lyophilizate contains about 5.5% to 6.0% water(Karl-Fischer procedure).

The invention will be illustrated in more detail by the followingnon-limiting example.

EXAMPLE

A bulk solution was prepared containing 30 mg/ml cyclophosphamide and 30mg/ml lactose. 10ml of the solution was placed in 100 20ml vials(nominal). The vials were placed in a lyophilization chamber. As shownin FIG. 1, the temperature was rapidly reduced to -30° C. and held atthis temperature for 4 to 5 hours until the solution was completelyfrozen. The condenser in the chamber was activated, the chamber wasevacuated to 500 microns. The vials were held at this condition 1 hourafter which the shelf temperature was raised to 0° C. The vials wereheld for 10 hours. As seen in FIG. 2 in this time, the producttemperature reached about -20° C. About 60 to 70% of the water wasremoved from the vial. At this time, the first stage lyophilizationprocess was stopped, the vacuum and condenser were turned off, chamberpressure was restored to atmospheric pressure and the shelf temperaturewas allowed to increase to 25° C. to melt the frozen cake andprecipitate the cyclophosphamide.

After 3-4 hours, precipitation was complete and the second stagelyophilization process was then initiated by rapidly freezing thesamples to -30° C. The samples were held for 5 hours until the solutionwas frozen. The condenser was activated and the pressure was reduced to500 microns. The vials were held for 1 hour and shelf temperature wasthen allowed to warm to 0° C. Two hours after the shelf temperaturereached 0° C., the product temperature reached 0° C. and the process wascompleted. The lyophilizate contained 5.5-6% water as determined byKarl-Fischer.

Fifty of the vials were placed on stability testing at 5° C. and 37° C.for six weeks. The results are shown in Table 1 below. To demonstratethe importance of retaining the cyclophosphamide as the monohydrate,fifty additional vials were also placed in a vacuum oven to drive offthe bound water and convert the monohydrate to the anhydrous form. Thesevials were also subjected to stability testing. The results are shown inTable 2. The results clearly show the superior stability of thelyophilizate prepared in accordance with the invention as compared toits anhydrous counterpart.

                  TABLE 1                                                         ______________________________________                                        Temp.   Time       Conc.    Avg.     Percent of                               (°C.)                                                                          (weeks)    (mg/vial)                                                                              (mg/vial)                                                                              Initial                                  ______________________________________                                        initial            294.4    294.4    100                                       5      6          299.3    299.3    102                                      37      6          294.9                                                                         288.4    291.6     99                                      ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Temp.   Time       Conc.    Avg.     Percent of                               (°C.)                                                                          (weeks)    (mg/vial)                                                                              (mg/vial)                                                                              Initial                                  ______________________________________                                        initial            294.3    294.3    100                                       5      6          300.1                                                                         300.0    300.6    102                                      37      6          141.5             48                                                          130.4             44                                                          247.7             84                                                          109.3             37                                       ______________________________________                                    

In summary, one manifestation of the present invention is a method forlyophilizing cyclophosphamide comprising the steps of freezing a bulksolution containing cyclophosphamide, a bulking agent, and water;removing a first portion of water from the frozen solution bylyophilization such that upon melting the solution, a solutionsupersaturated with cyclophosphamide is obtained; melting the solutionto produce said supersaturated solution; precipitating thecyclophosphamide from the solution as a hydrated polymorph; refreezingthe solution; and removing by lyophilization water not bound to thecyclophosphamide as a monohydrate or bound to the bulking agent.

In accordance with a preferred embodiment of the invention, the bulkingagent is an amorphous bulking agent and, more particularly, a bulkingagent selected from the group consisting of lactose, mannitol, glycineand valine. In a most preferred embodiment of the invention, the bulkingagent is lactose.

Preferably, the bulk solution contains about 3 to 3.3% cyclophosphamideand about 2 to 7% bulking agent and, more particularly, lactose.

The first stage lyophilization is initiated at a temperature lower than-15° C. and, most preferably, about -30° C. After cooling, the vials areheld for several hours to completely freeze them and then the pressureis reduced and the condenser is activated. Pressures up to 8000 micronscan be used but the preferred pressure is about 500 microns. The secondstage lyophilization conditions are similar or milder than the firststage.

Another manifestation of the invention is a pharmaceutical preparationcomprising a lyophilized cake including cyclophosphamide monohydrate andlactose. The lactose is present in an amount of about 0.3 to 1.7 partsper 1 part cyclophosphamide. Preferably, the lactose andcyclophosphamide are present in equal amounts and the cake containsabout 5.5 to 6% water. While not desiring to be bound, it is believedthat lyophilizates containing cyclophosphamide and lactose prepared bythe foregoing process have a unique crystal structure which renders themreadily soluble and improves chemical and physical stability.

Having described the invention in detail and by reference to preferredembodiments thereof, it will be apparent that modifications andvariations are possible without departing from the scope of theinvention defined in the appended claims.

What is claimed is:
 1. A pharmaceutical preparation consistingessentially of a lyophilized cake of cyclophosphamide monohydrate andlactose wherein said cake is prepared by the process offreezing a bulksolution containing cyclophosphamide, lactose, and water; removing afirst portion of the water from said frozen solution by lyophilization,such that upon melting said cake a solution supersaturated with saidcyclophosphamide and said lactose is obtained; melting said solution toproduce said supersaturated solution; precipitating saidcyclophosphamide as a hydrated polymorph; re-freezing said solution; andremoving by lyophilization water not bound to said cyclophosphamide orsaid lactose to yield a lyophilizate consisting essentially ofcyclophosphamide monohydrate and said lactose.
 2. The pharmaceuticalpreparation of claim 1 wherein, said bulk solution contains saidcyclophosphamide in an amount of about 3.0 to 3.3 percent and saidlactose in an amount of about 2 to 7 percent.
 3. The pharmaceuticalpreparation of claim 1 wherein said lactose is present in an amount ofabout 0.3 to 1.7 parts per 1 part said cyclophosphamide monohydrate. 4.The pharmaceutical preparation of claim 3 wherein said lactose and saidcyclophosphamide monohydrate are present in approximately equal amounts.